Rapid tests for the detection of E. coli are crucial in clinical laboratories and are important in monitoring the microbiological quality of food and water samples. The detection of E. coli and total coliforms in food and water samples is used as an indication of faecal contamination. The traditional methods used were based on membrane filtration and multiple-tube fermentation techniques. Increasingly in recent years, tests based on specific enzyme activities have been developed [A. Rompré et al, J. Microbial Methods, 49, 31-54, (2002)]. Several chromogenic and fluorogenic media have been developed that utilise β-D-glucuronidase activity as an indicator of E. coli and β-D-galactosidase activity for the simultaneous detection of total coliforms [J. Olstadt et al, J. Water and Health, 5, 287-282, (2007)]. These assays may be conducted in liquid media or on solid media such as agar plates. Some representatives among the many solid media commercially available that utilise this methodology serve as examples. C-EC Agar (Biolife Italiana Srl, Milan, Italy) uses fluorogenic MUG (4-methylumbelliferyl β-D-glucuronide) for the detection of E. coli and chromogenic X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) for the detection of total coliforms. The MI-agar reported by Brenner and colleagues [K. P. Brenner et al, Appl. Environ. Microbiol., 59, 3534-3544, (1993)] employs chromogenic indoxyl β-D-glucuronide for the detection of E. coli and the fluorogenic substrate MUGAL (4-methylumbelliferyl β-D-galactopyranoside) for total coliforms. Coliscan® (Micrology LLC, Goshen, USA), Chromocult® Coliform agar (Merck, Darmstadt, Germany) and Chromogenic Coliform Agar (Biolife Italiana Srl, Milan, Italy) adopt a dual chromogenic system with the substrates X-β-D-glucuronide (5-bromo-4-chloro-3-indolyl β-D-glucuronide) [dark-blue to violet colour with E. coli] and 6-chloro-3-indolyl β-D-galactopyranoside [red colonies with other coliforms]. It will be noted that all of the above-mentioned media contain indoxyl substrates. Indoxyl substrates work very well in solid media such as agar plates, but they are much less suited to liquid media, mainly because the indigo chromogen they produce is very insoluble. In the few examples of liquid media for detecting E. coli and total coliforms which do contain an indoxyl substrate, it is a galactoside for the visualisation of total coliforms. Thus, Fluorocult® LMX broth and Readycult® Coliforms 100 (Merck, Darmstadt, Germany) as well as LSB X-Gal MUG (Biolife Italiana Srl, Milan, Italy) all incorporate MUG for the detection of E. coli and X-Gal for the detection of coliforms. Several commercially important liquid media for the E. coli total coliforms application rely on substrates that are not based on indoxyl. Colitag® (CPI International, Santa Rosa. USA) and ColiLert® (Idexx Laboratories, Westbrook, USA) media both utilise MUG (blue fluorescence) for the detection of E. coli and ONPG (o-nitrophenyl-β-D-galactopyranoside) (yellow colour) for the detection of coliforms. The Colisure® assay (Idexx Laboratories, Westbrook, USA) uses MUG and chlorophenol red-β-D-galactopyranoside (magenta colour) for the same purpose.
All of the media containing MUG suffer from the practical disadvantage that a fluorescence detector or, at the very least, a UV lamp is required to visualise the fluorescent endpoint following the hydrolysis of this substrate. It would therefore be a great advantage to have a chromogenic substrate capable of detecting β-D-glucuronidase activity from viable bacteria that is suitable for inclusion in a liquid medium, especially for use in combination with a substrate for β-D-galactosidase activity like ONPG. It therefore follows that the chromogenic substrate should not hinder bacterial growth and it should be suitable for continuous assays. Among other desirable properties it should be affordable, easy to use, and give a colour that is readily distinguished even in the presence of a different chromogen resulting from the hydrolysis of a β-D-galactosidase substrate. None of the currently available enzyme substrates for β-D-glucuronidase intended for use primarily in liquid media are suitable. For instance, PNP-β-D-glucuronide gives the same yellow colour as ONPG. Phenolphthalein-β-D-glucuronide is very expensive and the endpoint can only be visualised by adding alkali to raise the pH, thus introducing an extra step as well as stopping bacterial growth. Resorufin-β-D-glucuronide is another extremely expensive substrate, as is fluorescein-di-β-D-glucuronide. Methyl esters of glucuronides have been used as substrates for β-D-glucuronidase activity. Fluorescein-di-β-D-glucuronide dimethyl ester is a product of Marker Gene Technologies, Inc. (Eugene, Oreg., USA) [catalogue number M0969] and has been advertised as a β-D-glucuronidase substrate since 2004 [Marker Gene Technologies Newsletter, Vol. 4, Nos. 9 and 10, 2004]. The manufacturers state that this substrate has enhanced cell-permeation properties in plants. They have also advocated the substrate for routine coliform detection [Marker Gene Technologies Newsletter, Vol. 5, No. 3, 2005]. Application WO2012/168415 claims some cost advantages to using lower alkyl esters of glucuronides as substrates in place of the free acids or their salts, and exemplifies this with the use of resorufin-β-D-glucuronide methyl ester. The application WO2012/168415 states that the alkyl esters are not in themselves efficient substrates for β-D-glucuronidase, but work because the free acids are formed by in situ hydrolysis. Thus the resorufin-β-D-glucuronide-6-methyl ester was hydrolysed by β-D-glucuronidase less efficiently than resorufin-β-D-glucuronide free acid, especially in the absence of added esterase. Moreover, the core molecule resorufin is still very expensive. Chlorophenol red-β-D-glucuronide has been obtained in very poor yield only [G. G. Y. Shen et al, U.S. Pat. No. 6,534,637 (2003)]. This substrate was not incorporated into bacterial growth media; therefore its suitability to detect E. coli and total coliforms remains unknown. However, when Brenner and colleagues [K. P. Brenner et al, Appl. Environ. Microbiol., 59, 3534-3544, (1993)] combined the related compound chlorophenol red-β-D-galactopyranoside with indoxyl β-D-glucuronide in a dual chromogenic assay for E. coli and total coliforms on an agar plate medium, they found that there was not enough colour contrast between the different colonies. Therefore, it is by no means certain that chlorophenol red-β-D-glucuronide would be an effective substrate in this application in liquid or tube bacterial growth media, especially in combination with a different chromogenic substrate.
Mention should also be made of another expensive glucuronide substrate of limited commercial availability that has been used to detect E. coli, namely 8-hydroxyquinoline-β-D-glucuronide. When this substrate is cleaved the aglycone forms a highly insoluble intense black chelate with iron compounds [A. L. James and P. Yeoman, Zentralbl. Bakteriol. Mikrobiol. Hyg. A, 267, 188, (1987)]. Although the substrate has been demonstrated in an agar plate medium [R. D. Reinders et al, Left. Appl. Microbiol., 30, 411-414, (2000)], toxicity of the aglycone to Gram-positive microbes has been observed [J. D. Perry et al, J. Appl. Microbiol., 102, 410-415, (2007)]. The substrate is not suitable for liquid media.
Enzyme substrates based on catechol have been described in EP1438423. However, this invention is restricted to chromogenic substrates giving a non-diffusible endpoint on solid media such as agar plates. Catechol-β-D-glucuronide is not disclosed in EP1438423. Although catechol-β-D-glucuronide has been mentioned occasionally in the scientific literature as a putative metabolic by-product from glucuronidation of either catechol or phenol, both the complete chemical synthesis and the characterisation of this glucuronide have yet to be described.
Bollenback and co-workers [G. N. Bollenback et al, J. Am. Chem. Soc., 77, 3310, (1955)] did prepare the fully protected precursor, catechol-2′,3′,4′-tetra-O-acetyl-β-D-glucuronide-6′-methyl ester, by chemical synthesis, but did not report deprotection of it to the free glycoside.
Maitra and Bhowmik [S. Bhowmik and U. Maitra, Chem. Commun., 2012, 46, 4624-4626] reported the use of 2,3-dihydroxynaphthalene-β-D-glucoside (DHN-β-D-glucoside) with terbium(III) in a luminescent assay for the detection of purified β-glucosidase. They make no mention of detecting microorganisms or of other glycoside derivatives of DHN.
Costa at al in Arch. Toxicol. (1999) 73:301-306 investigate metabolites derived from environmental benzene pollutant. The methodology subjects urine from rats fed with benzene to contact with β-glucuronidase, followed by analysis of the hydrolysis products by HPLC. Catechol is one of the hydrolysis products. The presence of a catechol glucuronide in the urine is assumed by inference. Costa et al do not isolate or characterise the catechol glucuronide. Costa at al do not provide any supporting evidence for the supporting structure, nor is there evidence for it being a a free glucuronide as opposed to an ester; esters are potential substrates for β-D-glucuronidase. Nor is there any evidence that the analyte that was hydrolysed was a mono-glucuronide, as opposed to a di-glucuronide. The use of β-glucuronidase to prove the structure of an analyte that is a substrate for the enzyme acknowledged in the art to be limited due to the lack of specificity of the enzyme. Thus β-glucuronidase hydrolyses other glycosides (Levvy at al in Glucuronic Acid Free and Combined, Ed. G J Sutton Academic Press New York and London 1966, page 317), so identifying a hydrolysis product is not conclusive proof that the starting substrate was even a glucuronide.
van der Hooft et al. in Analytical Chemistry (2012) 84(16) 7263-7271 identified pyrogallol-2-O-glucuronide as one compound present in quantities of less than 80 μmol in a complex mixture of metabolites in human urine after green tea intake. It was identified by mass spectrometry and 1H-NMR spectra. As reported by van der Hooft, the structure of pyrogallol-2-O-glucuronide is ambiguous as the authors do not state whether the compound is the α- or the β-isomer.